| Abstract |
Introduction
Mucormycosis is an emerging fungal disease among immunocompromised patients with hematological malignancies, undergoing transplantations or chronic steroid treatment, associated with high mortality, which can reach up tp 90% in disseminated disease. Unlike other filamentous fungi Mucorales can infect patents with uncontrolled hyperglycemia, diabetic ketoacidosis and iron overload, as well as immunocompetent individuals suffering from severe burns or trauma. The epidemiology of mucormycosis is attributed to Mucorales unique virulent traits, regarding spores size, iron acquisition and angioinvasion. Several studies have shown that Mucorales is resistant to killing by phagocytes, without revealing the related mechanisms. The aim of this dissertation was to shed light in the mechanisms regarding the interaction of macrophages with Mucorales spores and address the role of iron in the pathogenesis of mucormycosis.
Materials and Methods
All animal studies were performed with GFP-LC3, C57BL/6 (B6) or CD11cDTR mice were maintained in grouped cages in a high-efficiency particulate air-filtered environmentally controlled virus-free facility.
Aspergillus fumigatus ATCC46645 and Rhizopus strains used (WT R.oryzae ATCC55796932; WT strain R. delemar 99-880) were grown on Yeast extract agar glucose agar plates. In addition, Rhizopus FTR1 and FOB1/2 mutants were delivered from the aforementioned R.delemar strain. Fungal melanin was extracted from A. fumigatus and R. oryzae conidia, which were treated with a combination of proteolytic and glycohydrolytic enzymes, denaturing guanidine thiocyanate, and hot concentrated HCl. Rhizopus melanin was further characterized with liquid chromatography - mass spectrometry and electron paramagnetic resonance.
Bone marrow derived macrophages (BMDMs) were generated by culturing bone marrow cells obtained from 8- to 12-week-old female mice in culture medium supplemented with L929 cell-conditioned medium. For immunofluorescence experiments, BMDMs were stimulated with conidia of Rhizopus or A. fumigatus for the indicated time points. After infection, cells were fixed on the coverslips, which were then incubated with the indicated primary antibody (Ab), washed twice in PBS-BSA, then counterstained with the appropriate secondary Alexa Fluor secondary Ab. Images were acquired using a laser-scanning spectral confocal microscope (TCS SP2; Leica), LCS Lite software (Leica), and a ×40 Apochromat 1.25 NA oil objective using identical gain settings.
For the killing assays, 106 BMDMs were infected with either R.oryzae or A. fumigatus conidia, at an MOI of 1:1. At the indicated time point of infection (2 or 6 hours), BMDMs were lysed by sonication, centrifuged and the pellet containing intracellular conidia was resuspended in sterile PBS. Aspergillus fumigatus killing was assessed with confocal microscopy using propidium iodide staining, whereas killing of R. oryzae was assessed by photonic microscopy evaluating the germination of intracellular Rhizopus conidia.
For virulence studies, 8- to 12-week-old female mice were intratracheally infected with a standard dose of A. fumigatus or Rhizopus conidia and euthanized at the indicated time point. The lungs were homogenized, and CFU counts were assessed. For alveolar macrophages depletion studies, mice received by intratracheal administration 100 μl of clodronate liposomes or control liposomes. For CD11c cell depletion, CD11c-DTR mice received by intratracheal administration 20 ng/kg of diphtheria toxin (DT). The efficiency of cell depletion was assessed by immunohistochemistry for CD11c and flow cytometry analysis of bronchoalveolar lavage.
For the histopathological studies, lungs were fixed in 10% formalin, paraffin embedded, cut in 4-μm sections, and stained with hematoxylin and eosin. For immunohistochemistry studies, the anti-CD11c Abs and anti-CD68 primary antibodies were used for detection of CD11c and CD68 in tissue.
RNA was isolated both from BMDMs and R.oryzae spores. Briefly, obtained from 12-week-old female C57BL/6 mice were infected with R. delemar. At the indicated time point of infection (1, 4, and 18 hours), BMDMs were lysed using the RNeasy Plant Mini Kit (Qiagen). Afterwards, isolation of RNAs was performed according to the manufacturer’s instructions.
Results
In this study we showed that the alveolar macrophages play a predominant role during the host-Rhizopus interplay. Rhizopus conidia are phagocytosed by the alveolar macrophages of immunocompetent mice and establish prolonged intracellular dormancy, despite their in vitro vulnerability to the oxidative and non-oxidative killing mechanisms of the macrophages. Mechanistically, the resistance in killing is attributed to the melanin-induced phagosome maturation arrest. The alveolar macrophages selective depletion was associated with increased mortality and fungal burden in vivo experiments in immunocompetent mice, proving the essential role of macrophages on the natural course of mucormycosis. Furthermore, we discovered that inhibition of Rhizopus growth inside macrophages is a central host defense mechanism that depends on nutritional immunity via iron starvation. Finally, dual RNA sequencing (RNA-seq) and functional studies identify critical host and fungal modulators of iron homeostasis inside macrophages that promote invasive fungal growth. Discussion
In this dissertation, we showed the essential role of macrophages for the outcome of mucormycosis. Furthermore, our experiments introduce the prolonged intracellular survival of the fungus inside these immune cells as a central pathogenetic event in development of the infection. In addition, we dissect the molecular mechanisms that allow Rhizopus to persist inside macrophages via melanin-induced phagosome maturation arrest. Finally, we identify nutritional immunity via iron restriction inside the phagosome as an important host defense mechanism during pulmonary mucormycosis. These findings lead to a novel pathogenetic model of mucormycosis that links abnormalities in iron metabolism with nutritional immunity inside macrophages, paving the way for important future therapeutic implications in the management of this devastating disease
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